Despite the dramatic impact of rationally-developed, single-agent, 'targeted'therapy with the tyrosine kinase inhibitor imatinib (GleevecTM) in chronic myelogenous leukemia (CML), resistance to targeted therapies is still a significant clinical problem for patients with advanced phase CML and Ph+ ALL, a related but genetically- distinct BCR-ABL-dependent, high-risk lymphoid leukemia. This proposal describes a plan to develop cell-based assays for the systematic identification of drugs and small molecules that synergize with dasatinib, an FDA-approved, second generation, targeted BCR-ABL kinase inhibitor. In this approach, we seek to adapt our well characterized, robust pre-clinical murine leukemia model system to develop methodologies for the rational design of truly synergistic combination therapies. One of the underlying principles is that synergistic agents have different cellular target(s) and mechanism(s) of action, often acquire alternate mechanism of drug resistance, have distinct pharmacokinetic, pharmacodynamic and side-effect profiles, and yet dramatically enhance each other's therapeutic impact. In the context of leukemia therapy, we propose that synergistic drug combinations dramatically enhance initial eradication of leukemic cells, which directly reduces the pool of incipient drug-resistant cells to subsequently emerge during the course of continuous therapy. The central goal of this proposal is to further develop and configure cell-based assays for High-Throughput Screening (HTS) studies, with the goal of identifying known FDA-approved drugs and novel small molecules that will both overcome cytokine-dependent 'protection'and synergize with dasatinib to efficiently induce killing of engineered leukemia cells. Specifically, by incorporating our unique insights of cytokine-dependent, cell- extrinsic drug resistance into our innovative assay design, we aim to characterize existing drugs and discover novel small molecules that pheno-revert leukemogenic cells to their prior dasatinib-sensitive state. Conceptually, novel small molecules discovered in these studies represent unique pharmacological probes targeting pathways that may never have previously been implicated in the acquisition or maintenance of a drug-resistant phenotype. Our specific aims of this proposal include 1) the development of a cell-based, HTS- ready, primary screening assay that will reliably detect synergistic leukemic cell killing , and 2) The development of an efficient and systematic approach for evaluation, validation, and prioritization of active compounds identified in pilot and large-scale cell-based, synergy screening studies. PUBLIC HEALTH RELEVANCE: Despite the success with which modern chemotherapy is able to control many cancers, resistance of even a few residual cancer cells to therapy ultimately leads to clinical relapse and death of many cancer patients. Using well-defined experimental systems, we seek to develop reliable and robust methodologies to efficiently test or 'screen'thousands of existing drugs and new chemicals for their ability to effectively kill cancer cells.